In this issue

the recommended tests for diagnosing genetic causes of intellectual disability (ID), developmental delay (DD), and autism spectrum disorder (ASD). Current guidelines from the American College of Medical Genetics and Genomics (ACMG) and the American Academy of Pediatrics (AAP) suggest that children with ID, DD, and ASD receive FMR1 testing for fragile X syndrome (FXS) to determine repeats of the trinucleotide sequence CGG, plus chromosomal microarray (CMA) as fi rst-line tests. However, 2 signifi cant letters received by Genetics in Medicine suggest retiring routine FXS testing for ID, DD, and ASD because of low diagnostic yields. The fi rst letter, sent by geneticists at the University of California-Los Angeles (UCLA), University of California-Davis, and University of Washington points to unpublished research that shows a 0% yield for FXS testing at UCLA (Mullegama et al, 2017). The second letter points to a larger unpublished study at Children’s Hospital of Eastern Ontario (CHEO) in Ottawa, Canada, that also found a low yield (Hartley et al, 2017). Both letters were written in response to published research at Children’s National Medical Center in Washington, D.C., that found the yield for FXS testing for males with ID was 2.5% and 0% for boys with ASD (Weinstein et al, 2017). FXS testing “should be retired as a fi rst-tier test but remain part of the differential, particularly when well-defi ned features (physical and behavioral characteristics) and/or family history suggestive of fragile X syndrome are present,” says the letter concerning the low yield shown in UCLA testing results.

However, 2 signifi cant letters received by Genetics in Medicine suggest retiring routine FXS testing for ID, DD, and ASD because of low diagnostic yields. The fi rst letter, sent by geneticists at the University of California-Los Angeles (UCLA), University of California-Davis, and University of Washington points to unpublished research that shows a 0% yield for FXS testing at UCLA (Mullegama et al, 2017). The second letter points to a larger unpublished study at Children's Hospital of Eastern Ontario (CHEO) in Ottawa, Canada, that also found a low yield (Hartley et al, 2017).
Both letters were written in response to published research at Children's National Medical Center in Washington, D.C., that found the yield for FXS testing for males with ID was 2.5% and 0% for boys with ASD (Weinstein et al, 2017). FXS testing "should be retired as a fi rst-tier test but remain part of the differential, particularly when well-defi ned features (physical and behavioral characteristics) and/or family history suggestive of fragile X syndrome are present," says the letter concerning the low yield shown in UCLA testing results.

The Studies
The Children's National Medical Center study concludes that FXS testing as a fi rsttier test may not be necessary unless other criteria suggest the disorder. The analysis on 210 males with ID, learning disability (LD), or ASD who had CMA or FMR1 assays found that CMA detected abnormalities in 29% of males with ID/LD and 9% of males with ASD. In 80 males with ID/LD, just 2, or 2.5%, tested positive for FXS. Both patients had dysmorphic features and maternal family history. No males with ASD had positive FXS test results.
The UCLA study involved a retrospective analysis of 654 males who received testing between January 2002 and March 2017. The researchers excluded males tested for ataxia with no ID, DD, or autism. The FXS testing yield was 0%. The letter does note that males with classic symptoms or family history could have been excluded from the cohort by early diagnosis. It also points to studies from academic and commercial labs that have reported diagnostic yield of whole-exome sequencing (WES) for ID, DD, and ASD at 10% to 41%. The authors of the letter propose that genome-wide tests, including WES, become recommended fi rst-tier tests.
The CHEO study involved analysis of laboratory and clinical information for all 1,177 children referred for FXS testing between 2010 and 2015. This information included referral reason, age, sex, and number of CGG repeats. Although the number of children with full mutations remained constant at between 0 and 4 diagnoses per year, the overall diagnostic yield decreased over time. The highest yield was 2.4% in 2011. The overall yield during the entire study period was 0.009% among a total of 1,177 children.
Increased screening does not result in increased diagnosis, the CHEO authors suggest, and they note that current guidelines are based on studies conducted during an era of limited testing options and fewer known genetic causes of ID. Today, with more than 1,000 known genes related to ID, genome-wide tests are more appealing tests than FMR1, the letter adds.
More than 10 years ago, FXS testing was considered high-yield, says Julian A. Martinez-Agosto, MD, PhD, Associate Professor of Pediatrics and Human Genetics at UCLA and corresponding author of the letter concerning the study at the university. Studies informing current guidelines may have selected for classic symptoms of FXS or family history, which would make test yield higher than what most clinics observe. Increased uptake of American College of Obstetricians and Gynecologists-and ACMG-recommended premutation carrier screening and prenatal screening, in conjunction with termination of FXS-affected pregnancies, might drive the observed yield, he adds.

Reaction
The study institutions' relative proximity to dedicated FXS clinics may also contribute to low diagnostic yield, says Randi Hagerman, MD, Medical Director of the University of California (UC), Davis MIND Institute and Distinguished Professor and Endowed Chair in Fragile X Research in the Department of Pediatrics at the UC Davis School of Medicine in Sacramento. "It's too premature to say don't do [routine FXS] testing based on fi gures from clinics that don't have fragile X centers," Dr. Hagerman maintains. She points out that obstetricians often refer women with family history of FXS, early ovarian failure, or fertility problems to these centers.
Moreover, incidence of FXS varies between countries and regions of the United States. Dr. Hagerman points to a 2012 review article noting that prevalence estimates from U.S. studies range from 1 in 3,717 to 1 in 5,161 (Peprah, 2012).
Although the authors of letters about the UCLA and CHEO studies propose consideration of CMA and WES as fi rst-tier tests for ID, DD, and autism, an author of ACMG 2013 guidelines on FXS testing believes it is premature to recommend routine WES. "We need more data about exome sequencing's ability to identify individuals with FXS," says Elaine Spector, PhD, Director of the Molecular Genetics Laboratory at Children's Hospital Colorado in Denver in Aurora.
Dr. Spector and other guideline authors point out that next-generation sequencing technologies such as WES may not identify children with FXS because triplet repeat expansions like CGG are diffi cult to fi nd with the shortread alignment involved in determining sequences' locations on the genome. Currently, most molecular genetics laboratories cannot run and analyze WES assays, they add. FXS testing is cheaper and faster than WES, which is too complex for most labs, adds Dr. Hagerman. She points out that insurers readily cover the recommended FXS tests but not WES.
For now, Dr. Martinez-Agosto recommends following standard of care as established by ACMG guidelines. However, he suggests that geneticists track new data on WES and FXS testing yield. "Keep yourself updated on the rapidly changing fi eld of genetic testing," he advises.

Test results changed medical management for more than half of diagnosed babies
A recent study shows that whole-exome sequencing (WES) diagnoses in more than one-third of intensive care unit (ICU) infants with suspected single-gene conditions led to changes in medical management for more than half of babies with identifi ed disorders.
A retrospective study of 278 severely ill infants sequenced at Texas Children's Hospital in Houston between late 2011 and early 2017 reports that 36.7% of the infants received diagnoses leading to changes in management for almost 52% of those babies. In close to 55% of all infants, WES was a fi rst-tier diagnostic test (Meng et al, 2017).
The study is the latest to show the diagnostic power of WES, which has consistently yielded diagnoses in at least 25% of genetics patients with a variety of disorders.
"Whether it is redirection of care for a life-threatening condition or being able to offer lifesaving measures such as stem cell or organ transplant, exome sequencing promises a personalized approach to management of genetic diseases," says senior author Seema Lalani, MD, Associate Professor of Molecular and Human Genetics at Baylor University in Houston. She hopes the fi ndings will spur 8

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improved insurance coverage and wider use of WES.

The Study
The researchers studied seriously ill infants who received clinical exome sequencing within the fi rst 100 days of life. Subjects included 176 babies who had individual sequencing (proband-only sequencing), 39 infants tested as part of parent-child trios (trio testing), and 63 more critically ill babies who had more rapid trio sequencing (critical exome trio testing). Critical exome trio testing for these 63 infants was informed by clinical assessments by geneticists, with results delivered in an average of 13 days. WES led to diagnoses of 106 conditions in 102 babies. Six infants received a partial diagnosis, one of which was delivered by Southern blot analysis, or a nongenetic disorder. Of the remaining 170 infants who received no diagnoses from WES, 150 had chromosomal microarray. Microarray yielded no diagnoses. Critical trio exome sequencing provided the highest diagnostic yield, 50.8%, compared to 32.4% for proband-only sequencing and 33.3% for trio testing.
For 52% of patients overall, diagnoses spurred new subspecialist care, change in medication or diet, major procedures like transplant, or a switch from curative to palliative care. For patients who had proband-only testing, diagnoses yielded management changes for 45.6% of patients. That fi gure was 33.3% for trio testing patients. It was highest, 71.9%, for children diagnosed by critical trio exome testing.
Despite these results, 30, or 29.4%, of the 102 infants diagnosed by WES died before reaching an age of 120 days, the researchers note.
This study exposes several singlegene disorders that have been underascertained in critically ill newborns, says Dr. Lalani. For example, multiple babies in the study had Kabuki syndrome, and many had recently discovered disorders, such as those caused by the gene PURA, discovered in 2014. PURA variations can cause low muscle tone, respiratory problems, and feeding diffi culties. The researchers also saw several rare disorders, often with atypical presentations, that are diffi cult to diagnose by clinical exams of newborns, Dr. Lalani adds.

Reaction
The paper shows that babies in ICUs often have genetic conditions, says David Bick, MD, Chief Medical Offi cer, Hudson Alpha Institute for Biotechnology in Huntsville, Alabama, and former medical director of the genetics clinic at Children's Hospital of Wisconsin in Milwaukee. The institution was an early adopter of genomic sequencing, which can help doctors administer appropriate drugs to newborns in a timely fashion, Dr. Bick notes. He points to ALDH7A mutations that cause pyridoxine-responsive epilepsy, which should be treated as early as possible. "The more seizures a baby with this disorder has, the more damage there is to the brain. So pyridoxine is less effective with time," he explains.
The paper also demonstrates that sequencing can determine when treatment will not help an infant, he adds. "There are a ton of tricks we can do in the NICU that can keep kids alive. But we may put a child through a lot of pain for no gain," Dr. Bick says. But even rapid sequencing with 13-day turnaround "isn't fast enough for some kids." Sequencing helps diagnose serious disorders that are nonlethal in the neonatal period, when clinical features may be subtle or undetectable, notes Dr. Bick. He gives as examples RASopathies like Costello syndrome, cardiofaciocutaneous syndrome, and Noonan syndrome. Sometimes premature babies with Down syndrome will not have the telltale facial features until more than a month after birth, he adds.
Although the study's more rapid sequencing delivered favorable results, it may be diffi cult for many hospitals to perform, notes Jonathan S. Berg, MD, PhD, Assistant Professor in the Department of Genetics at the University of North Carolina at Chapel Hill, who is studying use of genomic sequencing in newborn screening. He points to the logistics and expense of having someone always available to receive samples, start lab work immediately, and speed up informatics procedures.
The clinical impact of genomic sequencing in the ICU needs further study, Dr. Berg adds. "We shouldn't overinterpret the results of this paper," he says, pointing to its retrospective design and focus on a very selected group of patients from specialized ICUs. This design, plus inclusion of patients from regular, neonatal, and cardiac ICUs, leaves the study open to confounding factors and limits researchers' ability to do a direct comparison of WES and microarray diagnostic yield, he explains.
Even so, this research and similar studies, especially controlled, prospective ones, can show insurance companies that genomic sequencing is really "just a drop in the bucket relative to the entire ICU stay," even with the higher cost of sequencing relative to other tests, says Dr. Berg. "If establishing a diagnosis can help to adjust the management for some fraction of patients and hopefully improve the outcomes for some of those, then perhaps genomic sequencing would be considered a worthwhile expense," he adds.
Overall, Dr. Bick is enthusiastic about the paper and believes clinical WES is no longer the test of the future. "For the most part, the day of using exome as a fi rstline test has arrived," he says. "But you will still need someone with expertise in dysmorphology to make the best use of it."

FRAGILE X SYNDROME PATIENT LACKS TYPICAL CGG REPEAT EXPANSION
Children who meet clinical criteria for fragile X syndrome (FXS) but whose test results do not show CGG repeats may have an atypical mutation in FMR1, write Sitzmann et al (p. 11, DOI: 10.1002/ ajmg.a.38504). The researchers describe an FXS patient with a rare FMR1 mutation previously reported in a single patient.
FXS is the most common inherited form of intellectual disability and is usually caused by FMR1 CGG-repeat expansions that prevent the gene's expression.
The 10-year-old child had a missense FMR1 mutation at c.413G>A but not the typical CGG-repeat expansion. He had classic FXS characteristics including intellectual disability, craniofacial findings, hyperextensibility, fleshy hands, flat feet, unsteady gait, and seizures. The child also had autism and attention deficit hyperactivity disorder. These are more FXS features than those of the previously reported patient.
A literature review revealed that 20 individuals with rare missense or nonsense mutations or other coding disturbances of the FMR1 gene ranged in age from infancy to 50 years. Their mutations included deletions, splicing errors, missense, and nonsense mutations. All reported individuals had intellectual disability; most were verbal with limited speech and had autism and hyperactivity.
Four of the 20 individuals had a mutation within exon 15, 3 within exon 5, and 2 within exon 2.
The researchers suggest that children with FXS features but no CGG-repeat expansion receive other tests, including nextgeneration sequencing, to rule out FXS.

PARENTS' EDUCATION INFLUENCES IQS OF CHILDREN WITH DOWN SYNDROME
A relatively high proportion of children with congenital limb defi ciencies need thorough evaluations because they also have other anomalies, write Bedard et al (p. 18, DOI: 10.1002/ajmg.a.38513).
The researchers analyzed Alberta (Canada) Congenital Anomalies Surveillance System data to determine both distribution of associated anomalies among congenital limb defi ciency cases and malformation patterns for syndrome classifi cation. The analysis included data on live births and stillbirths between 1980 and 2012 as well as statistics on early fetal deaths and terminations of pregnancies from 1997 to 2012 that involved a child or fetus with a congenital limb defi ciency and at least one major congenital anomaly.
Of 170 cases identifi ed, 75.3% were live born, 61.8% were male, 78.8% had longitudinal limb defi ciencies, and 77.6% had associated anomalies outside the musculoskeletal system. The researchers found signifi cant associations between preaxial limb defects and central nervous, gastrointestinal, and cardiovascular system anomalies. Associations between postaxial longitudinal defects and congenital hip and foot abnormalities were also observed.
The researchers suggest possible and probable syndrome diagnoses for cases with recognized malformation patterns, including tibial agenesis, humero-radial synostosis, neural tube disorders and limb defi ciencies, holoprosencephaly and limb defects, and other defects possibly caused by teratogens such as cigarettes, alcohol, and marijuana.
As next-generation sequencing becomes more mainstream, the proportion of cases with unrecognized patterns of malformations will decrease because more will have had a genetic diagnosis, the researchers predict. They suggest that surveillance systems incorporate these diagnoses based on unrecognized patterns of malformation to aid accurate classifi cation of cases. Recent research shows that variability among DS phenotypes is infl uenced by parental traits. In response, the researchers examined longitudinally the role of parental education as a proxy for parental intelligence as well as that education's infl uence on cognitive ability, measured at different points in development.
The researchers studied 43 children with DS who were 4 to 21 years of age and their parents. They determined mean education of both parents and measured their children's IQ with the Stanford-Binet 4th edition test at baseline and again 24 months later. The researchers divided the children into 2 age groups: 4 to 13 years of age for group 1 and 13 to 21 years of age for group 2.
The correlations between parental education and children's IQ scores were higher for group 2, with a mean r score of 0.46 versus a mean r score of 0.33 for group 2. Correlations between parental education and children's IQ increased over time, increasing from an r score of 0.26 to 39 for group 1 and from 0.49 to 46 for group 2.
These fi ndings and previous research can inform expectations for how children with DS will develop, say researchers.